WO1998002176A1 - Inhibition of signal transduction by the binding of non-phosphorylated compositions - Google Patents
Inhibition of signal transduction by the binding of non-phosphorylated compositions Download PDFInfo
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- WO1998002176A1 WO1998002176A1 PCT/US1997/012501 US9712501W WO9802176A1 WO 1998002176 A1 WO1998002176 A1 WO 1998002176A1 US 9712501 W US9712501 W US 9712501W WO 9802176 A1 WO9802176 A1 WO 9802176A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
Definitions
- the present invention is directed to a new class of SH2 domain binding peptides.
- the tyrosines of these peptides are not modified by phosphates or other similarly charged groups. Molecules that mimic this peptide structure may also bind to the Src Homology 2 (SH2) domain and inhibit its function.
- SH2 Src Homology 2
- the inhibition of signal transduction using inhibitors of critical interactions has important potential utility in the development of pharmaceutical agents.
- a problem in this field is the identification of novel lead structures that cause such inhibition.
- Several approaches have been suggested for the identification of such lead structures.
- One approach is determining the structure of the proteins involved and their naturally occurring binding targets or substrates. Then the relevant regions are modelled and synthetic analogues are generated. This is a difficult, expensive, time consuming and uncertain process.
- a second approach is screening random naturally occurring compounds for a desired activity. This approach requires significant specialized resources. In addition, the resulting compounds may or may not be amenable to structure activity relationship studies necessary to improve activity.
- linear phosphopeptides have been studied to identify those that bind to SH2 domains.
- the sequence specificity of those peptides may be indicative of the sequence consensus that allows for binding to the SH2 domain.
- these studies are often not particularly useful for the identification of structures that improve drug discovery.
- the linear peptides can adopt numerous three dimensional structures that complicate the generation of structural analogues .
- the peptides contained a phosphotyrosine .
- the equivalent non-phosphorylated peptides bound with reduced affinity.
- studies of the p85 SH2 domain the non-phosphorylated peptides bound with greatly reduced affinity.
- Phosphopeptides are highly charged and thus are often impermeable to cells. Modifications have been made that reduce the charge of the phosphotyrosine residue. However, no study has been reported that describes a peptide having an unmodified tyrosine residue as a competitor of a natural target.
- Grb2 is an example of a well established signal transduction protein in many cell control systems. Schlessinger, J.,- "SH2/SH3 signaling proteins," Curr . Opin. Genet. Dev. , 4: 25-30, 1994, incorporated herein in its entirety by reference.
- the natural targets for Grb2 include SHC phosphoprotein.
- a SHC phosphopeptide sequence that binds to the Grb2 SH2 domain, as well as other phosphopeptide sequences that bind to the Grb2 SH2 domain, are shown in Figure 1. From these sequences, a consensus sequence that binds to the Grb2 SH2 domain was determined.
- a consensus binding sequence was also determined experimentally by binding peptide mixtures to Grb2 , as described in Songyang, Z., et al . , "Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav, " Mol . Cell. Biol . , 14: 2777-2785, 1994, incorporated herein in its entirety by reference.
- the present invention is directed to a non-phosphorylated peptide or a functionally equivalent composition that binds to an SH2 domain of a signal transduction protein with high affinity. That is, the composition binds to the SH2 domain with an affinity similar to or greater than that of at least one phosphorylated peptide that is a natural target for the protein. Because the non-phosphorylated peptide or functionally equivalent composition binds to an SH2 domain with an affinity similar to or greater than a natural target, it can be used to inhibit the binding of the natural target .
- the present invention is also directed to a non-phosphorylated peptide or a functionally equivalent composition that binds to the Grb2 SH2 domain with high affinity. That is, the composition binds to the Grb2 SH2 domain with an affinity similar to or greater than that of a phosphorylated peptide derived from the SHC protein, which has a sequence DDPSpYVNVQ (SEQ ID NO: 1) .
- the present invention is directed to a non-phosphorylated peptide Gl comprising the following sequence : Cys-Glu-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys (SEQ ID NO: 2), -wherein the sequence has a disulfide linkage between the terminal cysteines. These flanking cysteines cause the formation of a disulfide constrained cyclic structure.
- the present invention is directed to a functionally equivalent peptide sequence or composition that binds to Grb2 SH2 domain with similar affinity as Gl .
- the present invention is also directed to a phage containing the above sequence or a functionally equivalent sequence as a variable region.
- the present invention is also directed to the inhibition of a signal transduction process, particularly those of the Grb2 SH2 domain, by administering a non-phosphorylated peptide or a functionally equivalent composition of the present invention.
- the present invention is directed to a process for forming a drug to block the inhibition of a signal transduction process that uses a non-phosphorylated peptide or a functionally equivalent composition, particularly Gl or a functionally equivalent sequence, as a model.
- Figure 1 demonstrates phosphopeptide binding sequences for Grb2 SH2 domains.
- Figure 1 also demonstrates a consensus sequence for the phosphopeptide binding sequences and compares these sequences to the Gl sequence .
- Figure 2A is a graph demonstrating that Gl phage binds to intact Grb2.
- Figure 2B is a graph demonstrating the specificity of Gl phage for Grb2.
- Figure 3A is a graph demonstrating Grb2 binding to phage Gl .
- Figure 3B is a graph demonstrating Grb2 binding to SHC phosphopeptide.
- Figure 4 is a graph demonstrating the poor binding of anti -phosphotyrosine antibodies to Gl phage.
- Figure 5 is a graph demonstrating the inhibition of Grb2 binding to SHC phosphopeptide.
- Figure 6 is a graph demonstrating the alteration of binding caused by the presence of various concentrations of different peptides.
- Figure 7 is a graph demonstrating the requirement of an YXN sequence in the Gl peptide.
- Figures 8A-C demonstrate the inhibition of complexes formed by the Grb2 SH2 domain in cellular extracts by G1TE peptide.
- Figure 8A demonstrates the structure of Gl and the G1TE molecule containing a thio- ether linkage.
- Figure 8B demonstrates the inhibition of Grb2 binding to the SHC phosphopeptide using surface plasmon resonance methods.
- Figure 8C demonstrates that the addition of G1TE and SHC phosphopeptide to lysates of cells inhibits the immunoprecipitation of complexes between Grb2 and pl85 erbB'2 . The binding of Grb2 to SOS1 is unaffected by G1TE peptide addition.
- the present invention discloses a non-phosphorylated peptide sequence, Gl, that binds to the Grb2 SH2 domain with similar affinity as compared to the SHC phosphoprotein.
- Gl binds to and blocks the Grb2 SH2 domain.
- the Gl peptide sequence apparently conserves only one position of the primary sequence of linear phosphopeptides shown to bind preferentially to the Grb2 SH2 domain. That is, Gl contains an asparagine at position +2 to the tyrosine.
- the Gl peptide demonstrates the principle that a small molecule can bind to the SH2 domain of Grb2 and inhibit its ability to bind phosphotyrosine containing proteins. Further, the Gl peptide demonstrates that non-phosphorylated peptides can bind to SH2 domains with high affinity, comparable to the binding affinity of phosphorylated peptides.
- the Gl peptide can be used as a starting point for the identification of other non-phosphorylated peptides that may bind to the SH2 domain with even greater affinity.
- the Gl peptide can be used as a starting point for the identification of functional groups on a small molecule that are critical for binding to the SH2 domain.
- the Gl peptide can be used as a structural model for non-peptide molecules with SH2 domain binding activity that may be identified by screening databases of compounds for chemically similar structures. Such database screening is generally described, for example, in Sun, E. and Cohen, F.E., "Computer-assisted drug discovery--a review," Gene, 137: 127-132, 1993, and Wang, S., et al .
- Gl peptide can be used as a basis for synthesis of peptide mimetic compounds. Olson, G.L., et al . , "Concepts and progress in the development of peptide mimetics," J. Med. Chem. , 36: 3039-3049, 1993, which is incorporated herein in its entirety by reference, teaches general principles of the process of synthesis of nonpeptide compounds that retain the biological activity of a peptide. The reference also describes some specific examples of replacement of peptide backbones.
- the Gl peptide serves as a model for drugs with significant advantages. Inhibition of SH2 domain functions have previously been attempted using structures that contain a phosphotyrosine or mimic it with a similarly charged group.
- the Gl peptide structure does not contain a phosphotyrosine or a similarly charged group on the tyrosine.
- Gl should offer improved cell penetration. Therefore, the Gl peptide sequence has significant advantages in that it and its derivatives may enter cells more efficiently than molecules based on the phosphopeptide.
- Gilmer, T. et al . "Peptide inhibitors of src SH3-SH2-phosphoprotein interactions," J. Biol . Chem. , 269: 31711-31719, 1994, which is incorporated herein in its entirety by reference, describes an unsuccessful experiment in which a phosphopeptide AcpYEEIE was used to attempt to block cell proliferation.
- Analogues of the Gl peptide may have the advantages of other small molecular weight pharmaceuticals including tissue penetration, bioavailability and ease of manufacture.
- the present invention describes a peptide sequence that can serve as a model for peptide and/or non-peptide pharmaceuticals.
- the Gl peptide may be used to find other peptide or non-peptide compounds that have similar structural elements that can bind to the Grb2 SH2 domain and block its signal transduction function. These compounds could have activity as drugs, for example, for the treatment of cancer or other hyperproliferative medical conditions.
- a measure of affinity of a peptide for the SH2 domain of a protein is the equilibrium dissociation constant, or K d .
- K d equilibrium dissociation constant
- a lower K d measured in terms of olarity, is indicative of greater affinity.
- Affinity of Grb2 for SHC phosphopeptide has been estimated by using surface plasmon resonance. Pelicci, G., et al . , "The motogenic and mitogenic responses to HGF are amplified by the She adaptor protein," Oncogene, 10: 1631-1638, 1995, incorporated herein by reference. Binding of SH2 domains of Grb2 to SHC phosphopeptide bound to a surface suggests a K d of 15 nM.
- a peptide- having affinity for the SH2 domain can be used to inhibit the binding of a natural target of the SH2 domain.
- IC 50 measures the concentration at which the peptides cause 50% inhibition of the natural target.
- a lower IC 50 measured in terms of molarity, is indicative of greater inhibition and thus greater affinity.
- the present invention is directed to a non-phosphorylated peptide or a functionally equivalent composition that binds to an SH2 domain of a signal transduction protein with high affinity. That is, the peptide or composition of the present invention binds to the SH2 domain with an affinity similar to or greater than that of at least one phosphorylated peptide that is a natural target for the protein. In particular, the peptide or composition of the present invention binds to the SH2 domain with an affinity for the SH2 domain greater than or within fifty fold less than the affinity of a phosphorylated natural target . Preferably, the affinity of the peptide or composition is greater than or within twenty-five fold less, and more preferably greater than or within fifteen fold less, than the affinity of a natural target.
- the peptide or composition has a K d and/or an IC B0 for an SH 2 domain less than or within fifty fold greater and preferably within twenty-five fold greater, and more preferably within fifteen fold greater, than that of at least one natural target of the SH 2 domain.
- non-phosphorylated peptide refers to a peptide that contains a tyrosine residue that has not been modified by a phosphate or a similarly charged group, such as a phosphorate or a malonate.
- similarly charged group is meant a group having a negative charge at normal intracellular pH and/or a group that is highly polar.
- Highly polar group may include groups that are at least as or more polar than oxygen.
- Highly polar groups may also include groups that are more polar than nitrogen.
- the "non-phosphorylated peptide" contains an unmodified tyrosine.
- the phrase "functionally equivalent composition” refers to a composition that is structurally based on the tertiary structure of at least a surface of a peptide that competitively binds to a signal transduction protein and that retains the function of that peptide as a competitive binder of the signal transduction protein.
- tertiary structure includes the charge distribution, as well as the polarity and the ability to hydrogen bond, of the peptide.
- the phrase "natural target” refers to phosphopeptides that are naturally occurring in vivo and bind with the SH2 domain of a signal transduction protein with sufficient affinity such that a signal is produced.
- the natural target is the natural target having the greatest affinity for the SH2 domain of a particular signal transduction protein.
- the present invention is also directed to a non-phosphorylated peptide or a functional equivalent composition that binds to the Grb2 SH2 domain with high affinity, that is, a peptide or composition that binds to the Grb2 SH2 domain with an affinity similar to that of a phosphorylated peptide derived from the SHC protein having a sequence DDPSpYVNVQ (SEQ ID NO: 1) .
- This sequence of the SHC protein is a known target for the Grb2 SH2 domain.
- Rozakis-Adcock, et al . "Association of the She and Grb2/Sem5 SH2 -containing proteins is implicated in activation of the Ras pathway by tyrosine kinases," Nature, 360: 689-692, 1992, incorporated herein in its entirety by reference.
- the affinity of the peptide or functionally equivalent composition for the Grb2 SH2 domain is greater than or within fifty fold less than the affinity of the SHC protein having SEQ ID NO: 1.
- the affinity of the composition is less than or within twenty- five fold less, and more preferably within fifteen fold less, than the affinity of the SHC protein.
- the peptide or functionally equivalent composition has a K d and/or an IC 50 for an Grb2 SH 2 domain less than or within fifty fold greater and preferably within twenty-five fold greater, and more preferably within fifteen fold greater, than that of the SHC protein.
- the present invention is also directed to a non- phosphorylated peptide comprising sequence Gl or to a functionally equivalent composition that binds to Grb2 SH2 domain with similar affinity.
- Functionally equivalent compositions include functionally equivalent peptide sequences as well as non-peptide functionally equivalent compositions .
- the phrase "functionally equivalent peptide sequence” or “functionally equivalent sequence” refers to a non-phosphorylated peptide sequence in which one or more modifications have been made that do not significantly affect the function of the sequence as a competitive binder of a signal transduction protein.
- alterations in the peptide that substitute amino acids at one or more positions may be possible and still retain functional equivalence.
- Such substitutions may include replacing the tyrosyl group with a functionally equivalent group, such as a phenyl or other aromatic group.
- Such modifications do not include the addition of a phosphate or a similarly charged group to the tyrosine or a functionally equivalent residue.
- the functionally equivalent sequence or functionally equivalent peptide sequence of the present invention has an affinity for a Grb2 SH2 domain greater than or within fifty fold less, and preferably within twenty-five fold less, and more preferably within- fifteen fold less, than the affinity of the SHC protein having SEQ ID NO: 1.
- the functionally equivalent sequence has a K d and/or an IC 50 for a Grb2 SH 2 domain less than or within fifty fold greater and preferably within twenty-five fold greater, and more preferably within fifteen fold greater, than that of the SHC protein.
- Gl and its functionally equivalent sequences and/or compositions may be used alone or as part of a biologically entity or a synthetic structure, which may include a phage.
- the insertion of a sequence into a phage is within the knowledge of one of ordinary skill in the art.
- Gl may be inserted into a fd phage, such as fUSE5.
- the composition may also be part of a fusion protein, such as with Gst.
- the present invention is also directed to the inhibition of a signal transduction process, particularly those of the Grb2 SH2 domain, by administering a non-phosphorylated peptide or functionally equivalent composition as discussed above.
- the peptide or functionally equivalent composition of the present invention may be administered by any known method, including oral and parenteral administration.
- the peptide or functionally equivalent composition may be administered alone or with a pharmaceutically acceptable carrier. Dosages may be routinely determined and varied to obtain the desired effect .
- the present invention is directed to a process for forming a drug to block the inhibition of a signal transduction process that uses a non-phosphorylated peptide or functional equivalent composition, particularly Gl or a functionally equivalent sequence or composition, as a model. That is, the present invention is directed to processes in which Gl or a functionally equivalent sequence or composition is used as a basis for creating new drugs that block the inhibition of a signal transduction process.
- Methods for the conversion of a peptide into a non-peptide mimetic compound include: use of non L-amino acids or other amino acid analogues in synthesis, determination of the structure of a peptide and use of this structure to guide synthesis of similar non-peptide molecules, and use of the special positioning of essential chemical groups in a peptide to search databases for compounds of similar structure.
- the Gl peptide sequence was identified using phage display techniques similar to those taught in U.S. Patent No. 5,223,409, to Ladner et al . , which is incorporated herein in its entirety by reference.
- a library was constructed to contain more than 10 7 different peptide sequences bounded by cysteine residues inserted into the Gene III protein of the fd phage fUSE5.
- a variation of standard methodology for purifying specific phages bound to a target protein from among the large diversity of the library was used. Scott, J.K. and Smith, G.P., "Searching for peptide ligands with an epitope library, " Science, 249: 386-390, 1990, incorporated herein in its entirety by reference .
- the Grb2 protein was generated as a glutathione S-transferase (GST) fusion protein using recombinant expression vectors in E. coli as taught in Sastry, et al . , "Quantitative analysis of Grb2-Sosl interactions: the N-terminal SH3 domain Grb2 mediates affinity, " Oncoqene , 11:1107-12, 1995, incorporated herein in its entirety by reference.
- the phage library was allowed to bind to the recombinant Grb2 protein.
- the GST-Grb2 proteins were collected on glutathione sepharose . Unbound phages were removed by washing and bound phages were eluted. These Grb2 binding phages where allowed to infect E. coli and the amplified phages were isolated. Phage binding to GST bound to glutathione sepharose were removed. This affinity isolation binding to Grb2 was repeated twice.
- phages Following the third Grb2 binding step, eighteen resulting phages were isolated. The isolated phages were subjected to nucleotide sequencing. The variable region of each phage showed an identical nucleotide sequence and predicted protein sequence. The predicted sequence is that of Gl . Variations on the above phage selection procedure may yield other phages either related to the Gl peptide or of a different sequence class.
- Gl phage extracts were prepared by inoculating a culture containing 40 ⁇ g/ml tetracycline with an active tetracycline resistant phage containing bacterial colony and growing overnight . Phage stocks were allowed to bind to the immobilized target proteins for two hours at room temperature. The plates were then washed twice with wash buffer. Sheep anti-phage antibody was incubated in a blocking buffer at 1:1000 dilution for one hour at room temperature. Donkey anti-sheep antibody coupled to alkaline phosphatase was incubated with the plates (1:1000 dilution) for one hour at room temperature. The plates were washed twice with wash buffer. Then the color was developed using a p-nitro-phenyl phosphate (PNP) substrate . As seen in Figure 2A, only the full length
- the concentration dependence of binding of GST-Grb2 to immobilized phage targets was measured.
- the concentration of GST-Grb2 was varied in a binding reaction and the extent of bound GST-Grb2 was measured using anti-GST antibodies.
- the binding of GST-Grb2 to Gl phage was compared with the binding of GST-Grb2 to a phosphorylated peptide derived from the SHC protein (sequence: DDPSpYVNVO) , a known target of Grb2 SH2 domain, in vivo .
- target protein Gl phage or SHC phosphopeptide was bound to polystyrene 96 well microtiter plates at 1 ⁇ g/well in PBS buffer for 16 hours at 4°C.
- the Gl phage was prepared by PEG precipitation of stationary phage bacterial cultures of Gl infected E. coli.
- the SHC phosphopeptide target was prepared by crosslinking the phosphopeptide to Bovine Serum Albumin (BSA) by a l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDO using standard procedures, as taught in Harlow, E. and Lane, D. , Antibodies: A Laboratory Manual .
- BSA Bovine Serum Albumin
- EEO l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
- GST-Grb2 protein was prepared as described in Sastry, et al . , "Quantitative analysis of Grb2-Sosl interactions: the N-terminal SH3 domain Grb2 mediates affinity," Oncogene , 11:1107-12, 1995, incorporated herein in its entirety by reference.
- GST-Grb2 was incubated in the plate wells for two hours at room temperature. Non-specific binding reactions were inhibited using 5% BSA. Phosphatases were inhibited by 1 mM NaV0 4 in the binding buffers. The plates were washed twice with wash buffer.
- Binding was detected using sequential reactions of anti-GST antibody antisera (Santa Cruz Bioche ) followed by goat anti-mouse IgG antibody coupled to alkaline phosphatase followed by calorimetric development using PNP substrate.
- anti-GST monoclonal antibody was incubated in blocking buffer at lng/ml for one hour at room temperature.
- goat anti-mouse IgG antibody antisera coupled to alkaline phosphatase was incubated with the plates (1:1000 dilution) for one hour at room temperature. The plates were washed twice with wash buffer. Then the color was developed using a PNP substrate.
- target protein Gl phage or SHC phosphopeptide was bound to polystyrene 96 well microtiter plates at 1 ⁇ g/well in PBS buffer for 16 hours at 4°C.
- the Gl phage was prepared by PEG precipitation of stationary phage bacterial cultures of Gl infected E. coli.
- the SHC phosphopeptide target was prepared by EDC crosslinking to BSA as described above.
- the plates were then blocked with blocking buffer containing 5% BSA, lOmM Tris pH 7.5, 150mM NaCl (TBS) and ImM NaV0 4 .
- the plates were then washed three times with wash buffer containing 0.1% BSA in TBS.
- the GST-Grb2 protein was prepared as previously described above. GST-Grb2 (0.5 ⁇ M) was incubated in plate wells for two hours at room temperature. Monoclonal anti -phosphotyrosine antibody (Santa Cruz Biochemicals) was incubated with the plates at lng/ml for two hours at room temperature. The plates were washed twice with wash buffer. Anti-GST monoclonal antibody (Santa Cruz Biochem) was incubated in blocking buffer at lng/ml for one hour at room temperature. Goat anti-mouse antibody coupled to alkaline phosphatase was incubated with the plates (1:1000 dilution) for one hour at room temperature. The plates were washed twice with wash buffer and color was developed using a PNP substrate.
- target protein Gl phage or SHC phosphopeptide was bound to polystyrene 96 well microtiter plates at 1 ⁇ g/well in PBS buffer for 16 hours at 4°C.
- the Gl phage was prepared by PEG precipitation of stationary phage bacterial cultures of Gl infected E. coli.
- the SHC phosphopeptide target was prepared by EDC crosslinking to BSA using standard procedure as described above. The plates were then blocked with blocking buffer containing 5% BSA, lO M Tris pH 7.5, 150mM NaCl (TBS) and ImM NaV0 4 . The plates were then washed three times with wash buffer containing 0.1% BSA in TBS.
- the GST-Grb2 protein was prepared as described above. GST-Grb2, at a concentration of 0.5 ⁇ M, was preincubated in binding buffer with various concentrations of soluble SHC phosphopeptide for two hours and allowed to bind to the plate bound targets. The plates were washed twice with wash buffer. Anti-GST monoclonal antibody (Santa Cruz Biochem) was incubated in blocking buffer at lng/ml for 1 hour at room temperature. Goat anti-mouse antibody coupled to alkaline phosphatase was incubated with the plates (1:1000 dilution) for one hour at room temperature. The plates were washed twice with wash buffer and color was developed using a PNP substrate. As shown in Figure 5, the concentrations of
- the peptide was chemically synthesized and a form containing the cysteine residues in oxidized, disulfide bonded form was isolated. Standard methods were used for solid phase chemical synthesis of the peptide. The oxidized, disulfide bonded peptide formed spontaneously during the isolation of the peptide. The formation of this conformation was confirmed by mass spectrometric analysis, indicating a molecular weight of 1321. In addition, 10 mM DTT induced alteration in mobility in C18 chromatography experiments using an acetonitrile gradient in 0.5% trifluoroacetic acid. These experiments demonstrate an altered conformation on reduction and therefore confirm the presence of the disulfide bond.
- the Gl peptide (CELYENVGMYC [disulfide bonded] ) was used as an inhibitor of the interaction of the Grb2 with a SHC phosphopeptide in surface plasmon resonance (Biacore) experiments.
- the SHC phosphopeptide was attached to the solid surface by incorporation of a biotin at the N-terminus as described in Pelicci, G. , et al . , "The motogenic and mitogenic responses to HGF are amplified by the She adaptor protein," Oncogene, 10: 1631-1638, 1995, incorporated herein by reference.
- This biotinylated phospho-SHC peptide was then bound to the streptavidin on the surface of SA5 chips (Pharmacia- Biacore) .
- the binding was conducted at a 2nM and 5 ⁇ l/minute for 10 minutes.
- the peptides were mixed with 200 nM GST-Grb2 in HBS buffer (Pharmacia Biacore) and allowed to bind for 10 minutes at a flow rate of 5 ⁇ l/minute.
- the Ru change at equilibrium was calculated and the percent of control (no added peptide) binding was calculated.
- the Gl peptide was compared to SHC phosphopeptide
- DDPSpYVNVQ G1C-S
- SELYENVGMYS SHC control peptide
- the results are shown in Figure 6.
- the G1C-S peptide demonstrates that the disulfide bond may be required.
- the conformational constraint imparted by the disulfide bond in the Gl peptide may be required.
- the SHC control demonstrates the poor affinity of the linear non-phosphorylated SHC peptide.
- G1.4Y-A contains an alanine substitution in place of the tyrosine at position 4 (CELAENVGMYC) and G16N-A contains an alanine substitution for the asparagine at position 6 (CELYEAVGMYC) .
- No inhibition of Grb2 binding to the SHC phosphopeptide was seen with either peptide at a concentration of 50 ⁇ M.
- the binding of peptide to the Grb2 SH2 domain was measured by surface plasmon resonance (Pharmacia Biacore) . The percent inhibition was calculated by comparison of the Ru eq in the presence of peptide with the Ru eq with no inhibiting peptide present.
- cyclic peptides used in the above examples were formed by disulfide or thio-ether bonds but could be formed by other chemical linkages. Direct cyclization of peptide sequences using a peptide bond in place of the two cysteine residuals or with intervening amino acid is possible. Peptides of constrained structure could also contain cysteines separated by 4-12 amino acids. Peptides could also contain non-natural amino acids such as D amino acids. Modifications in peptide bonds and other peptide mimetic structures are possible. Furthermore, not all of the amino acids of the reported sequence may be necessary for activity. Peptides with single or multiple conservative substitutions are likely to be active.
- Peptides where non-conservative alterations do not change the overall three dimensional conformation of the peptide are likely to be active in a manner similar to the Gl peptide.
- the Gl peptide is likely to contain 2-8 functional groups that interact with the Grb2 SH2 domain. The three dimensional position of these groups may be mimicked by synthetic or natural products. Such compounds may also have activity as SH2 antagonists, and are encompassed by the present invention.
- the present invention thus demonstrates that SH2 domains can be inhibited by non-phosphorylated structures in which the tyrosine has not been modified by a phospho or a similarly charged group. Therefore other SH2 domain interactions of other proteins, such as SHC, crk, nek, ragGAP, P13kinase, PLC gamma, src, or others, may also have non-phosphorylated inhibitors . This is in contrast to efforts to mimic the phosphotyrosine with other modifications or blocking groups.
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EP97936092A EP0969854B1 (en) | 1996-07-16 | 1997-07-16 | Inhibition of signal transduction by the binding of non-phosphorylated compositions |
DE69735208T DE69735208D1 (en) | 1996-07-16 | 1997-07-16 | SUPPRESSION OF SIGNAL TRANSMISSION BY BINDING OF NON-PHOSPHORYLATED COMPOSITIONS |
AU38847/97A AU3884797A (en) | 1996-07-16 | 1997-07-16 | Inhibition of signal transduction by the binding of non-phosphorylated compositions |
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US2185896P | 1996-07-16 | 1996-07-16 | |
US60/021,858 | 1996-07-16 |
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Cited By (4)
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WO2000073326A2 (en) * | 1999-06-02 | 2000-12-07 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Redox-stable, non-phosphorylated cyclic peptide inhibitors of sh2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
US6951915B2 (en) | 1999-06-02 | 2005-10-04 | The United States Of America As Represented By The Department Of Health And Human Services | Redox-stable, non-phosphorylated cyclic peptide inhibitors of SH2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
ITMI20081742A1 (en) * | 2008-09-30 | 2010-04-01 | Multimedica Holding S P A | USE OF PEPTIDES IN ANTIANGIOGENIC THERAPY RELATED TO CANCER PATHOLOGIES |
ITMI20081743A1 (en) * | 2008-09-30 | 2010-04-01 | Multimedica Holding S P A | USE OF PEPTIDES IN ANTIANGIOGENIC THERAPY |
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Publication number | Priority date | Publication date | Assignee | Title |
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AU4972096A (en) * | 1995-02-01 | 1996-08-21 | Affymax Technologies N.V. | Peptides and compounds that bind to sh2 domains |
-
1997
- 1997-07-16 WO PCT/US1997/012501 patent/WO1998002176A1/en active IP Right Grant
- 1997-07-16 EP EP97936092A patent/EP0969854B1/en not_active Expired - Lifetime
- 1997-07-16 DE DE69735208T patent/DE69735208D1/en not_active Expired - Lifetime
- 1997-07-16 AU AU38847/97A patent/AU3884797A/en not_active Abandoned
Non-Patent Citations (6)
Title |
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FELDER ET AL.: "SH2 Domains Exhibit High-Affinity Binding to Tyrosine-Phosphorylated Peptides Yet Also Exhibit Rapid Dissociation and Exchange", MOLECULAR AND CELLULAR BIOLOGY, vol. 13, no. 3, March 1993 (1993-03-01), pages 449 - 455 * |
ROJAS ET AL.: "An Alternative to Phosphotyrosine-Containing Motifs for Binding to an SH2 Domain", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 234, 29 May 1997 (1997-05-29), pages 675 - 680, XP000857070, DOI: doi:10.1006/bbrc.1997.6694 * |
SALCINI ET AL.: "Formation of Shc-Grb2 complexes is necessary to induce neoplastic transformation by overexpression of Shc proteins", ONCOGENE, vol. 9, 1994, pages 2827 - 2836 * |
SASTRY ET AL.: "Multple Grb2-Protein Complexes in Human Cancer Cells", INTERNATIONAL JOURNAL OF CANCER, vol. 70, no. 2, 17 January 1997 (1997-01-17), pages 208 - 213 * |
See also references of EP0969854A4 * |
SONGYANG ET AL.: "Specific Motifs Recognized by the SH2 Domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.", MOLECULAR AND CELLULAR BIOLOGY, vol. 14, no. 4, April 1994 (1994-04-01), pages 2777 - 2785, XP001095617 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000073326A2 (en) * | 1999-06-02 | 2000-12-07 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Redox-stable, non-phosphorylated cyclic peptide inhibitors of sh2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
WO2000073326A3 (en) * | 1999-06-02 | 2001-05-25 | Us Gov Health & Human Serv | Redox-stable, non-phosphorylated cyclic peptide inhibitors of sh2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
US6951915B2 (en) | 1999-06-02 | 2005-10-04 | The United States Of America As Represented By The Department Of Health And Human Services | Redox-stable, non-phosphorylated cyclic peptide inhibitors of SH2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
US7259142B2 (en) | 1999-06-02 | 2007-08-21 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Redox-stable, non-phosphorylated cyclic peptide inhibitors of SH2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
ITMI20081742A1 (en) * | 2008-09-30 | 2010-04-01 | Multimedica Holding S P A | USE OF PEPTIDES IN ANTIANGIOGENIC THERAPY RELATED TO CANCER PATHOLOGIES |
ITMI20081743A1 (en) * | 2008-09-30 | 2010-04-01 | Multimedica Holding S P A | USE OF PEPTIDES IN ANTIANGIOGENIC THERAPY |
Also Published As
Publication number | Publication date |
---|---|
EP0969854B1 (en) | 2006-02-01 |
AU3884797A (en) | 1998-02-09 |
EP0969854A4 (en) | 2002-12-04 |
EP0969854A1 (en) | 2000-01-12 |
DE69735208D1 (en) | 2006-04-13 |
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